Role of microRNA319 in creeping bentgrass salinity and drought stress response

Abstract

The microRNA319 family (miR319) is one of the most conserved and ancient microRNA (miRNA) families in plants. Transgenic creeping bentgrass (Agrostis stolonifera) overexpressing a rice miR319, Osa-miR319a, exhibited enhanced salt and drought tolerance. A comprehensive hypothetical model about the role of miR319 in creeping bentgrass response to salinity and drought stress was proposed. Salinity and drought stress induces elevated expression of miR319, resulting in downregulation of at least 4 putative target genes of miR319 (AsPCF5, AsPCF6, AsPCF8, and AsTCP14) as well as a homolog of the rice NAC domain gene AsNAC60, and therefore positively contributing to plant abiotic stress response. Hormones might also regulate miR319 and its targets, and the expression level of the miR319 targets might be a balance of miR319-mediated target cleavage and hormone regulation of the targets. Furthermore, HKT gene families involved in salt exclusion mechanisms as well as mechanisms controlling the timing of gene expression network are also hypothesized to play an important role in this pathway.

The microRNA319 (miR319) family, one of the most conserved and ancient microRNA (miRNA) family, has previously been found to be responsive to multiple stresses including salinity, drought, cold, and aluminum stress based on high-throughput sequencing and microarray profiling data.^1-6 In recent years, an increasing number of studies on how miR319 is involved in plant stress response were conducted to define the function of miR319. For example, a hypothetical model of how miR319 regulates plant cold stress response in sugarcane was proposed.⁵ In this model, miR319 was upregulated by both cold stress and phytohormone abscisic acid (ABA) treatment, which caused repression of its targets leading to inhibition of plant development. It was also hypothesized that ABA might induce miR319 targets, and that the final expression level of miR319 targets was a balance of miR319-mediated cleavage and ABA induction of the targets.⁵ Interestingly, a recent study in rice⁷ demonstrated that overexpression of miR319 led to enhanced plant cold tolerance (4 °C) after chilling acclimation (12 °C), and that the expression level of the five target genes of miR319 (OsPCF5, OsPCF6, OsPCF7, OsPCF8, and OsTCP21) all decreased in transgenic plants. It was further demonstrated that downregulation of either one of the 2 target genes, OsPCF5 or OsPCF8 via RNA interference (RNAi) also resulted in enhanced cold tolerance in transgenic plants.⁷ We have investigated the role of miR319 in plant abiotic stress resistance using transgenic creeping bentgrass (Agrostis stolonifera) and found that miR319 regulates plant response to drought and salinity stress via downregulation of its target genes.⁸

Cold stress causes mechanical damages, while salinity and drought stresses mainly impose osmotic and ionic pressure on plant cells.⁹ In nature, plants usually have to combat with a combination of multiple stresses instead of a single one, which requires crosstalk of various signals and coordination of different stress-responsive pathways.⁹ Based on the above-mentioned potential involvement of miR319 in diverse stressors, it is plausible to postulate that miR319 could be one of the most important master regulators involved in different stress-responsive pathways. Here we propose a hypothetical model of the molecular mechanisms of miR319-mediated plant salinity and drought stress tolerance in creeping bentgrass (Fig. 1).

Figure 1. Hypothetical model of molecular mechanisms of miR319-mediated plant salinity and drought stress tolerance in creeping bentgrass. The accumulation of mature miR319 was elevated upon salt and drought stress, which causes downregulation of at least 4 putative target genes of miR319 (AsPCF5, AsPCF6, AsPCF8, and AsTCP14) as well as a homolog of the rice NAC domain gene AsNAC60, and therefore positively contributing to plant abiotic stress response. Hormones might also regulate miR319 and its targets, and the expression level of the miR319 targets might be a balance of miR319-mediated target cleavage and hormone regulation of the targets. Furthermore, HKT gene families involved in salt exclusion mechanisms as well as mechanisms controlling the timing of gene expression network are also hypothesized to play an important role in this pathway.

In creeping bentgrass, the accumulation of mature miR319 was elevated upon salt and drought stress, which caused the downregulation of at least 4 putative target genes of miR319 (AsPCF5, AsPCF6, AsPCF8, and AsTCP14) (Fig. 1).⁸ These target genes were hypothesized to serve as negative regulators of plant abiotic stress tolerance and plant development. We also examined what other stress-related genes are involved in miR319-mediated stress tolerance pathway. A previous study analyzing stress-responsive genes in rice has revealed that a member of NAC family (NAM from petunia, ATAF and CUC from Arabidopsis), named ONAC60 (Os12 g41680) was significantly upregulated by salt stress.¹⁰ ONAC60 has been known to be the target gene of miR164 in rice,¹¹ and TCP genes have been shown to regulate miR164 in Arabidopsis.¹² Theoretically, if this mechanism is conserved across plant species, the expression level of AsNAC60, our newly identified ONAC60 homolog in creeping bentgrass, would be expected to increase in transgenic plants overexpressing miR319 via miR319 repression of TCP-mediated miR164 regulation pathway. Surprisingly, the expression level of AsNAC60 in transgenic creeping bentgrass plants was decreased (Fig. 1),⁸ suggesting that the regulation of NAC60 expression is species-dependent. It is also possible that AsNAC60 might be downregulated by other more powerful regulators. The functions of ONAC60 have not been fully characterized in model plant species rice, and remain to be investigated.

TCPs have been demonstrated to regulate jasmonic acid (JA) synthesis and leaf senescence in Arabidopsis,¹³ while miR319 was found to be responsive to ABA treatment in sugarcane,⁵ to positively regulate auxin (AUX) signals in rice and Arabidopsis,^14-16 and to be involved in coordinating the antagonistic functions of AUX and cytokinin (CK) pathways and gibberellic acid (GA) and ABA pathways.^14,17,18 Furthermore, analysis of upstream promoter sequence of miR319 in Arabidopsis has revealed the existence of stress-related elements including MBS (drought), HSE (heat stress), AREs (hypoxic, low temperature, and dehydration stress), TC-rich repeats (defense and stress), TGA element (auxin), AuxRR core (auxin), and TCA element (SA).² The hormones JA, salicylic acid (SA), AUX, and ABA have all been postulated or shown to play an important role directly or indirectly in plant response to abiotic stress.¹⁹ Thus, it is intriguing to assume that in creeping bentgrass, miR319 also played an important role in phytohormone crosstalk and abiotic stress signaling transduction. Additionally, the expression of miR319 and its targets might be subjected to regulations of several phytohormones or other regulators. The ultimate expression levels of these genes reflect the homeostasis equilibrium of multiple factors (Fig. 1).

Our results in creeping bentgrass suggested that transgenic plants overexpressing miR319 might adopt salt exclusion mechanisms to improve its salinity tolerance.⁸ Coincidently, in transgenic rice plants overexpressing miR319, the Na⁺ transporter gene OsHKT2 was upregulated (Yang C, Li D, Zhu L, personal communication). It would be interesting to investigate whether any expression changes in HKT gene families take place in transgenic creeping bentgrass overexpressing miR319. This would allow better understanding of the miR319-mediated salt exclusion mechanism (Fig. 1).

In rice, the more rapid response of Osa-miR319 genes and their targets to low temperature was associated with improved plant cold tolerance.⁷ In contrast, the expression change of sugarcane miR319 was delayed in cold tolerant cultivars.⁵ These data indicated that besides expression level changes in miR319 and their targets, timing was also critical for coordinated interactions of multiple regulators determining plant stress response. Further investigation in this direction would provide information to substantiate the current hypothetical model of miR319-mediated plant stress response pathway (Fig. 1).

Glossary

Abbreviations:

miRNA

microRNA

miR319

microRNA319

ABA

abscisic acid

AUX

auxin

CK

cytokinin

ET

ethylene

GA

gibberellic acid

JA

jasmonic acid

SA

salicylic acid

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by Biotechnology Risk Assessment Grant Program competitive grant no. 2007–33522–18489 and no. 2010–33522–21656 from the USDA National Institute of Food and Agriculture as well as the USDA grant CSREES SC-1700315 and SC-1700450. Technical Contribution No. 6219 of the Clemson University Experiment Station.